Detection of blood plasma amygdalin of dissipating blood stasis botanical

ABSTRACT

A detection method of blood plasma amygdalin of dissipating blood stasis botanical is disclosed. The method includes: (1) adding 2-5 mol/L phosphoric acid to plasma of mammalian administered dissipating blood stasis botanical and mixing the solution of plasma-phosphoric acid (1:2-3, v/v), applying the solution to small column of Waters Oasis HLB activated by methanol and water; after leaching by water and 80-100% methanol and eluting by 0.2-1% ammonia-methanol, drying and enriching the eluent at 25-30° C.; and redissolving with mobile phase; (2) UPLC/MS measuring: UPLC condition: chromatographic column: Acquity UPLC BEH C 18 , 2.1′100 mm, mobile phase A: water-acetonitrile-formic acid 95:5:0.1 v/v/v, mobile phase B: acetonitrile-formic acid 100:0.1 v/v; MS condition: electric spraying ion source (ESI), detecting with positive ion mode, scanning at the range of m/z 150-800. The method can be used for pharmacokinetics study of amygdalin of dissipating blood stasis botanical.

TECHNICAL FIELD

This invention belongs to the field of pharmacokinetics, particularlyinvolves the determination method of blood plasma Amygdalin ofstrengthening body resistance and dissipating blood stasis botanical(vegetable).

BACKGROUND ART

Strengthening body resistance and dissipating blood stasis botanical arecomposed of compound prescriptions including salvia miltiorrhiza, peachkernel, Schisandra chinens etc., which have the effect of curing liver,lung and kidney fibrosis; however, due to lack of pharmacokineticsresearch has been carried out on the strengthening body resistance anddissipating blood stasis botanical, so it is not clear about theeffective ingredients in vivo, and it is difficult to provide a basisfor quality control and guiding clinical rational administration, thushinder those drugs from entering the international market.

So far, no relevant pharmacokinetic research report on strengtheningbody resistance and dissipating blood stasis botanical has been found,and there is not any detection method of Amygdalin of the compoundprescription in biological samples (including blood plasma sample).

CONTENTS OF THE INVENTION

The technical matters aim to be resolved by this invention is to providea detection method of blood plasma Amygdalin of strengthening bodyresistance and dissipating blood stasis botanical (vegetable), and themethod is used for pharmacokinetics research, and clarifying thepharmacokinetics rules of the blood plasma Amygdalin of thestrengthening body resistance and dissipating blood stasis.

-   -   The technical matters solved by this invention are achieved        through the following technical solutions:    -   The detection method of blood plasma Amygdalin of strengthening        body resistance and dissipating blood stasis botanical        (vegetable) includes the following steps:    -   (1) Pretreatment of Mammalian Blood Plasma Samples

-   a. Collect mammal plasma containing drugs after being administered    the strengthening body resistance and dissipating blood stasis    botanical, misce bene after adding 2-5 mol/L phosphoric acid, and    the volume ratio between the blood plasma and phosphoric acid is    1:2-3, applying the blood plasma with drugs to small column of    Waters Oasis HLB activated by methanol and water; after leaching    with water and 80-100% methanol and eluting with 0.2-1%    ammonia-methanol, drying and enriching the eluent under the    condition of 25-30° C., and redissolving the eluent with mobile    phase.

-   b. Detection with UPLC-MS method;    -   (2). UPLC/MS Detection Method

UPLC condition: chromatographic column: Acquity UPLC BEH C18, 2.1×100mm,mobile phase A: water-Acetonitrile-Formic acid 95:5:0.1 v/v/v, mobilephase B: Acetomtrile-Formic acid 100: The electrospray ionization (ESI)ion source of the described step (2), detects with positive ion mode,the desolvation gas flow is 440 L/h, the desolvation gas temperature is300° C., the cone gas flow is 50 L/h, the ion source temperature is 100°C., the spray capillary voltage is 3800 V, the sampling cone voltage is30V, the extracting cone voltage is 2.00 V, the lens voltage is 0.1 V,and mass scanning at the range of m/z 150-800. In solid-phase extractionprocess of this invention, the adsorption capacity of the solid phase tothe analytes is greater than the sample mother liquor, when the samplespass through the solid-phase extraction column, the analytes and anumber of similar ingredients were adsorbed on the surface of solid, andother ingredients then pass through the column with the sample motherliquor, first leach columns with larger polar solvents, to rinse andremove a number of unrelated ingredients, and filially elute theanalytes with appropriate solvent, drain and enrich the eluent; use UPLCsystem, separate the analytes with other ingredients in the elution, andfinally detected with mass spectrometry detector.

The Amygdalin in this invention is water-soluble ingredients, usingsolid-phase extraction method, can fully extract the Amygdalin in thesamples, combined with using a UPLC/MS system to detect, markedlyimprove the resolutions of Amygdalin among other ingredients in thesamples, and the analysis method is more sensitive and faster, tofacilitate the detection of the blood plasma concentration of theAmygdalin in pharmacokinetic research.

DESCRIPTION OF FIGURES

FIG. 1. UPLC-MS chromatogram of Amygdalin.

MODE OF CARRYING OUT THE INVENTION

Combining with the specific embodiments, further elaboration of thisinvention is given below. It should be understood that these embodimentsare only for description of the present invention but not for the use oflimiting the scope of the present invention. It should also beunderstood, after reading the contents taught in this invention,technicians in this field can make various changes or modification tothis invention, these equivalent forms are all included in the scopedefined by the claims attached to this application.

Embodiment 1

A detection method of blood plasma Amygdalin of strengthening bodyresistance and dissipating blood stasis botanical (vegetable).

-   1. Method of pretreating mammalian blood plasma sample: Collect    mammal plasma containing drugs after being administered the    strengthening body resistance and dissipating blood stasis    botanical, mince bene after adding 2-5 mol/L phosphoric acid, and    the volume ratio between the biological sample and phosphoric acid    is 1:2-3, applying the blood plasma with drugs to small column of    Waters Oasis HLB activated by methanol and water; after leaching    with water and 80-100% methanol and eluting with 0.2-1%    ammonia-methanol, drying and enriching the eluent under the    condition of 25 to 30° C., and redissolving the eluent with mobile    phase.-   2. UPLC/MS determination method: The analysis conditions of the    applied UPLC/MS method in this invention, UPLC condition:    chromatographic column: Acquity UPLC BEH C18, 2.1×100 mm, mobile    phase A: Water-Acetonitrile-Formic acid 95:5:0.1 v/v/v, mobile phase    B: Acetomtrile-Formic acid 100:0.1 v/v, eluting in accordance with    the following gradient:

Flow Rate Time (min) (mL/min) % A % B 0 0.300 100 0 5.00 0.300 85.0 15.010.00 0.300 70.0 30.0 20.00 0.300 40.0 60.0 30.00 0.300 20.0 80.0 35.000.300 20.0 80.0 35.01 0.300 100.0 0.0 38.00 0.300 100.0 0.0

MS conditions: electrospray ionization(ESI) ion source, detecting withpositive ion mode, the desolvation gas flow is 440 L/h, the desolvationgas temperature is 300° C., the cone gas flow is 50 L/h, the ion sourcetemperature is 100° C., the spray capillary voltage is 3800 V, thesampling cone voltage is 30V, the extracting cone voltage is 2.00 V, thelens voltage is 0.1 V, and mass scanning at the range of m/z 150 to 800.

Detection results: Amygdalin can be detected in the blood plasma ofmammals after drenched with strengthening body resistance anddissipating blood stasis botanical (see FIG. 1).

1. The detection method of blood plasma Amygdalin of strengthening bodyresistance and dissipating blood stasis botanical includes the followingsteps: (1) Pretreatment of Mammalian Blood Plasma Samples a. Collectmammal plasma containing drugs after being administered thestrengthening body resistance and dissipating blood stasis botanical,misce bene after adding 2 to 5 mol/L phosphoric acid, and the volumeratio between the blood plasma and phosphoric acid is 1:2-3, applyingthe blood plasma with drugs to small column of Waters Oasis HLBactivated by methanol and water; after leaching with water and 80-100%methanol and eluting with 0.2-1% ammonia-methanol, drying and enrichingthe eluent under the condition of 25 to 30° C., and redissolving theeluent with mobile phase; b. Detection with UPLC/MS Method; (2) UPLC/MSdetection method. UPLC condition: chromatographic column: Acquity UPLCBEH C18, 2.1×100 mm, mobile phase A: water-Acetonitrile-Formic acid95:5:0.1 v/v/v, mobile phase B: Acetomtrile-Formic acid 100:0.1 v/v; MScondition: electrospray ionization (ESI) ion source, detecting withpositive ion mode, and mass scanning at the range of m/z 150-800.
 2. Thedetection method of blood plasma Amygdalin of strengthening bodyresistance and dissipating blood stasis botanical described inaccordance with claims 1, its features lie in: the described step (2)detects with positive ion mode, the desolvation gas flow is 440 L/h, thedesolvation gas temperature is 300° C., the cone gas flow is 50 L/h, theion source temperature is 100° C., the spray capillary voltage is 3800V, the sampling cone voltage is 30V, the extracting cone voltage is 2.00V, the lens voltage is 0.1 V, and mass scanning at the range of m/z 150to 800.